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Updated: Jun 10, 2025

In Vitro Analysis of E3 Ubiquitin Ligase Function
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In Vitro Analysis of E3 Ubiquitin Ligase Function

Published on: May 14, 2021

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Rapid turnover of CTLA4 is associated with a complex architecture of reversible ubiquitylation

Pei Yee Tey1, Almut Dufner2,3, Klaus-Peter Knobeloch2,3

  • 1Biochemistry, Cell and Systems Biology, Institute of Systems, Molecular and Integrative Biology, University of Liverpool , Liverpool, UK.

The Journal of Cell Biology
|October 15, 2024
PubMed

Insights

Cytotoxic T-lymphocyte-associated protein 4 (CTLA4) degradation is regulated by ubiquitylation and the deubiquitylase USP8. Loss of USP8 accelerates CTLA4 ubiquitylation and turnover, impacting its cellular levels and exosome secretion.

Area of Science:

  • Immunology
  • Cell Biology
  • Molecular Biology

Background:

  • Cytotoxic T-lymphocyte-associated protein 4 (CTLA4) is a key immune checkpoint regulator.
  • CTLA4 is a short-lived membrane protein, suggesting rapid degradation pathways.

Purpose of the Study:

  • To investigate the molecular mechanisms governing CTLA4 degradation.
  • To identify proteins involved in regulating CTLA4 stability and turnover.

Main Methods:

  • Ubiquitylation analysis at specific lysine residues (203 and 213).
  • v-ATPase inhibition and cycloheximide treatment to assess CTLA4 stability.
  • Exosome isolation and analysis.
  • Depletion and re-expression studies of USP8 and HD-PTP.
  • UbiCRest analysis of ubiquitin chain linkages.

Main Results:

  • CTLA4 degradation is dependent on ubiquitylation at K203 and K213.
  • USP8 interacts with CTLA4 and its loss enhances CTLA4 ubiquitylation and degradation.
  • HD-PTP depletion mimics USP8 loss effects on CTLA4 ubiquitylation.
  • CTLA4 is also secreted in exosomes, a process influenced by USP8.
  • UbiCRest identified mixed ubiquitin chain linkages (Lys63, Lys27, Lys29) on CTLA4.

Conclusions:

  • USP8 plays a critical role in regulating CTLA4 stability and degradation through ubiquitylation.
  • Distinct ubiquitin chain linkages may contribute to CTLA4's rapid turnover.
  • Understanding CTLA4 regulation is crucial for immunotherapy and immune response modulation.

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